Crop losses caused by Fusarium oxysporum is getting serious around the world. Medicago sativa having huge cultivation area is one of the crops affected from this fungal pathogen, so it is an important issue to farmers. In order to struggle diseases, plants have many mechanisms such as secondary metabolite production, and pathogenesis related protein synthesis including antimicrobial peptides (AMPs). AMPs are one the most important of them because they are specific for the pathogen types. In this study, it was reported that the assessment of the expression levels of MsDef1 and MsSN1 AMPs in M. sativa roots against F. oxysporum infection after pre-treatment of salycilic acid, CaO NPs and their combinations. MsDef1 and MsSN1 expression levels were significantly increased after foliar application of all SA concentration, 2 ppm CaO NPs and the combination of 1 ppm CaO NPs in 1 mM SA. The highest expression level was seen in 5 mM SA after 24 h of application. Also, it was examined antioxidant enzyme activities (CAT and SOD), and PPO and PAL enzyme, two important enzymes of the plant defense mechanism. PAL and PPO activities were slightly increased after the applications. According to our study, foliar application of SA and CaO NPs lead to significant increasing in the expression level of MsDef1 and MsSN1 against F. oxysporum, one of the most importat plant pathogens, which indicates the importance of the antimicrobial peptides in first line of defense in plants.